Heparan sulfate (HS) and chondroitin sulfate (CS) are glycosaminoglycans. Heparan sulfate and chondroitin sulfate modified proteoglycans have been shown to play an important role in regulating the function of a number of glycosaminoglycan (GAG) binding proteins which include growth factors and chemokines. For example, binding of basic fibroblast growth factor (b-FGF) to HS modified proteoglycans has been shown to increase local concentrations of the growth factor and prolong its half life. In addition, it appears that growth factor binding to the proteoglycan changes its conformation and facilitates its interaction with its receptor and also leads to receptor oligomerization enhancing signal transduction.
An example of a proteoglycan of current interest is CD44. CD44 is a widely distributed type I membrane protein which is capable of binding hyaluronan (HA), other extracellular matrix components and osteopontin. The interaction between CD44 and its ligands has been shown to participate in cell migration and activation. The exons encoding the CD44 gene can be variably spliced to give rise to multiple protein isoforms. Expression of these CD44 isoforms can be tissue specific, developmentally regulated and/or regulated by cell activation. Most of these isoforms arise from the variable splicing of exons encoding polypeptide fragments located in the extracellular region of CD44, downstream of the HA binding domain. Changes in the pattern of glycosylation of CD44 resulting from the addition of variably spliced exons, which are modified extensively with O-linked carbohydrates, can effectively modulate the HA binding activity of CD44. Likewise, different CD44 isoforms are modified with different GAG polymers including HS and CS.
The present invention concerns a method for enhancing the biological activity of a glycosaminoglycan binding protein comprising administering to a subject an effective amount of an artificial proteoglycan which is a recombinant fusion protein having a glycosaminoglycan assembly site comprising the sequence SG to which is bonded chondroitin sulfate or both chondroitin sulfate and heparan sulfate, wherein said recombinant fusion protein comprises:
(a) a first polypeptide which comprises a control sequence and the glycosaminoglycan assembly site wherein the control sequence and the assembly site result in modification of the polypeptide with chondroitin sulfate or both chondroitin sulfate and heparan sulfate, and
(b) a second targeting polypeptide.
In another aspect, the present invention is directed to a method for modifying a protein having a glycosaminoglycan assembly site comprising the sequence SG, said method comprising recombinantly inserting nucleic acid encoding a proteoglycan or fragment thereof containing the control sequence into an expression vector encoding said protein such that said protein is expressed as a fusion protein comprising said proteoglycan or fragment thereof, and when said fusion protein is expressed in a host cell, said fusion protein is modified with chondroitin sulfate or both heparan sulfate and chondroitin sulfate.
Other aspects of the invention are artificial proteoglyans and chimeric nucleic acids encoding artificial proteoglycans.